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In this research, a hydroxyl group functionalized metal-organic framework (MOF), UiO-66-(OH)2, was synthesized as a “on-off-on” fluorescent switching nanoprobe for highly sensitive and selective detection of Fe3+, ascorbic acid (AA) and acid phosphatase (ACP). UiO-66-(OH)2 emits yellow-green light under ultraviolet light, when Fe3+ was added, Fe3+ was chelated with hydroxyl group, the electrons in the excited state S1 of the MOF transferred to the half-filled 3d orbits of Fe3+, resulting in fluorescence quenching because of the nonradiative electron/hole recombination annihilation. AA could reduce Fe3+to Fe2+, which can destroy the electron transfer between UiO-66-(OH)2 and Fe3+after AA adding, resulted in nonoccurrence of the nonradiative electron transfer, leading to the recovery of UiO-66-(OH)2 fluorescence intensity. The probe can also be used to detect ACP based on the enzymolysis of 2-phospho-L-ascorbic acid (AAP) to produce AA. Benefitting from the hydroxyl group and the characteristics of UiO-66, including the high porosity and large surface area, the developed UiO-66-(OH)2 showed extensive advantages as a fluorescent probe for detection of multi-component, such as high sensitivity and selectivity, colorimetric detection, fast response kinetics and easy to operate, economical and secure. This is the first time to use active group functionalized MOFs as a multi-component sensor for these three substances detection.